Use of enzyme polymorphism and hybridization crosses to identify sibling species of the mosquito, Anopheles quadrimaculatus (Say)

USE OF ENZYME POLYMORPHISM AND HYBRIDIAZTION CROSSES TO IDENTIFY
SIBLING SPECIES OF THE MOSQUITO, Anopheles guadrimaculatus (Say)

By

GREGORY CHARLES LANZARO

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE

UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS

FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA
1986

THIS WORK IS DEDICATED TO THE MEMORY OF THE AUTHOR'S

FATHER, FRANK

ACKNOWLEDGEMENTS

The author wishes to express his sincere appreciation to Dr.
J. A. Seawright for his guidance and continued friendship
throughout the course of the work resulting in this paper.
Special thanks are extended to Dr. S. K. Narang for his
instruction in electrophoretic techniques and interpretation of
results. The author extends his gratitude to the graduate
committee members Drs. D. W. Hall and S. C. Schank for their
encouragement and critical review of the work presented.

Very special thanks are extended to S . E. Mitchell and P. E.
Kaiser for their support and friendship. Thanks are extended to
B. K. Birky, L. A. Dickinson and M. Q. Benedict for helping in
many ways with this effort. Finally, special thanks are extended
to Ms. R. C. Brewington for assistance in the preparation of this
manuscript.

TABLE OF CONTENTS

PAGE
ACKNOWLEDGEMENTS iii

ABSTRACT v

CHAPTER I. ISOZYME PHENOTYPES AND INHERITANCE PATTERNS

OF ENZYME VARIANTS IN Anopheles quadrimaculatus

(Say) 1

Introduction 1

Material and Methods 2

Results 14

Discussion 27

CHAPTER II. EXPERIMENTAL HYBRIDIZATION OF GEOGRAPHIC
STRAINS OF Anopheles quadrimaculatus

(Say) 28

Introduction 28

Materials and Methods 30

Results 35

Discussion 52

CHAPTER III. ENZYME POLYMORPHISM AND GENETIC STRUCTURE
OF POPULATIONS OF Anopheles quadrimaculatus

Species A and B 58

Introduction 58

Materials and Methods 59

Results 61

Discussion 77

CONCLUSIONS 85

BIBLIOGRAPHY 87

BIOGRAPHICAL SKETCH 92

Abstract of Dissertation Presented to the Graduate School of

the University of Florida in Partial Fulfillment of the

Requirements for the Degree of Doctor of Philosophy

USE OF ENZYME POLYMORPHISM AND HYBRIDIZATION CROSSES TO IDENTIFY
SIBLING SPECIES OF THE MOSQUITO Anopheles quadrimaculatus (Say)

By

GREGORY CHARLES LANZARO
December 198 6

Chairman: J. A. Seawright

Major Department: Entomology and Nematology

Work was conducted on the population genetics of the
mosquito. Anopheles quadrimaculatus (Say) . The research
consisted of three parts: 1) electrophoretic techniques and
enzyme phenotypes; 2) hybridization experiment; and 3) population
genetics. Techniques were developed to visualize twenty-seven
enzyme loci. The phenotypes of these are described and the
inheritance patterns of nine of the polymorphic loci presented.
Hybridization experiments were conducted to determine the mating
compatibilities of nine geographic populations. Hybrid sterility
in males produced from some of these crosses revealed the
existence of two sympatic sibling species of A . quadrimaculatus at
three of nine sites. Analysis of isozyme frequencies of twenty
loci, also confirmed the existence of the sibling species.
Genotypic frequencies of heterozygotes for alleles at two enzyme
loci, Idh-1 and Idh-2 , were significantly deficient for

heterozygotes at the same three localities identified in the
hybridization experiment. Heterozygote deficiency was also
observed at a fourth site not included in the hybridization
experiment. The IDH loci were identified as being diagnostic for
the two species and were used as a tool for assembling gene
frequency data into discrete populations of each. An analysis of
gene frequencies resulted in calculations of genetic distance
between the two species, tentatively designated A.
guadrimaculatus species A and B. The values obtained for genetic
distance were consistent with values previously published for
sibling species in the genus Anopheles.

CHAPTER I

ISOZYME PHENOTYPES AND INHERITANCE PATTERNS OF ENZYME

VARIANTS IN Anopheles quadrimaculatus (Say)

Introduction

The southern house mosquito. Anopheles quadrimaculatus
(Say) is one of the five species comprising the Nearctic
Anopheles maculipennis complex. Work on the genetics of
these species is limited. Salivary gland chromosomes have
been described and polytene maps have been prepared for all
the species (Kitzmiller, et al., 1967). Of the five species
in the group, A. quadrimaculatus has been most studied
genetically. The inheritance of DDT and dieldrin resistance
have been described (Davidson, 1963; French and Kitzmiller,
1964) in this species.

In addition the inheritance of a number of morphological
mutants have been described. These include stripe (French
and Kitzmiller, 1963) , red-stripe (Mitchell and Seawright,
1984b) , black body (Seawright and Anthony, 1972) and brown
body (Mitchell and Seawright, 1984a) .

The karyotype of this species is comprised of two
metacentric autosomes (chromosomes 2 and 3) and a pair of
heteromorphic sex chromosomes (Kitzmiller and French, 1961) .

Recently the mutants brown-body and stripe were assigned to
chromosomes 2 and 3, respectively (Mitchell and Seawright,
1984a) .

In the present study electrophoretic techniques were
developed for the visualization of twenty enzyme systems
representing twenty-seven enzyme gene loci. The inheritance
patterns for nine of the polymorphic loci are presented.
This study provides the groundwork for mapping studies using
enzyme genes and a tool for the analysis of the population
genetics of this species.

Materials and Methods
Gels were made using three parts Connaught starch
(Connaught Laboratories Limited, Willowdale, Ontario, Canada)
and one part Electrostarch (Electrostarch Company, Otto-
Hiller, Madison, Wisconsin). A 12.5% (w/v) solution of the
starch mixture in the appropriate gel buffer was heated over
a gas flame in a 1000 ml Erlenmeyer filtration flask. The
mixture was continuously swirled by hand during the entire
cooking process, which generally took 4-5 minutes. When the
solution came to a vigorous boil the heating process was
terminated. The solution was immediately degassed by
attaching the sidearm of the flask to a vacuum line. A
vacuum was drawn over the solution until all small air
bubbles were removed. The gel solution was then poured into
a mold. The gel molds were constructed of 1/4 inch
plexiglass with the top horizontal surface measuring 20 cm x

12.6 cm X 1 cm. This surface held that portion of the gel
through which the samples migrated during electrophoresis.
Each of the long sides of the horizontal was connected to a
leg running perpendicularly, so that the entire surface
formed an inverted U shape. Each of the perpendicular side
walls was open along the bottom. The openings were closed
with two inch masking tape when the gel was poured. A volume
of 400 ml of buffer yielded a gel 1 cm thick. After pouring,
the gel was cooled for about two hours at room temperature,
then covered with saran wrap. The cast gels were further
cooled, for at least two hours prior to loading, in a
refrigerator at 5°C.

Samples were prepared for electrophoresis by first
making a crude homogenate of individual adult mosquitoes. A
block of 3/4 inch plexiglass containing sixty-four 1/4 inch
deep wells was used to hold samples for homogenization. Each
well was filled with thirty microliters of deionized water,
and the block was then wrapped in saran wrap and cooled in a
refrigerator for at least one hour. The block was placed in
a container of crushed ice, and an individual adult mosquito
was placed in each of thirty wells. The specimens were
homogenized by means of stainless steel rods, which were
attached to a brass plate in four rows of four rods per row.
These were positioned on the brass plate so that the wells in
the plexiglass block served as a template into which the
sixteen rods fit. By rocking the plate rapidly from side to
side sixteen samples could be homogenized simultaneously.

The homogenates were each absorbed onto 9 x 3 mm wicks
cut from Whatmann 3 MM filter paper. Thirty samples and
three bromphenol blue dye markers were inserted into an
incision in the gel at a position 2.5 cm from the cathodal
end.

Prior to loading the gels the electrode buffer chambers
were filled with the appropriate electrode buffer and placed
in the refrigerator. The chambers were rectangular boxes
measuring 23x7x4. 5 cm constructed of 1/4 inch plexiglass.
The chamber was partitioned by a divider into two subchambers
one 3.5 cm wide the other 2 cm wide. The smaller subchamber
contained the electrode (20 gauge platinum wire) which was
connected to a banana plug set in one end of the chamber.
The large subchamber provided a place for the leg of the gel
mold to be set. A set of two chambers, anode and cathode
completed the apparatus. Each chamber held 250 ml of
electrode buffer.

The loaded gel was readied for the electrophoretic run
by first removing the masking tape from the openings in the
legs of the gel mold and then setting each leg in an
electrode chamber. This arrangement allowed current to pass
through a continuous, U shaped gel so that no sponge or paper
was used to connect the gel to the electrode buffer.
Although this required using more starch, it provided a
superior connection, since sponge or paper connectors can
become dislodged or dry out.

The entire apparatus was placed in a refrigerator at 5°C
to keep the gel cool. In addition, the top surface of the
gel was covered with saran wrap and a plastic box containing
crushed ice was placed on top, for additional cooling.
Current, 125-250V, was applied to the gel by using an ISCO
regulated high voltage supply unit Model 493.

Three buffer systems were required for electrophoresis

of the enzymes included in this study. A description of the

buffers follows:

1. CA-8 Tris-Citrate (Steiner and Joslyn, 1979)

gel buffer: .074 M Tris (hydroxymethyl)

aminomethane (Tris)
.009 M citric acid
pH 8.45
none

1.37 M Tris
.314 M citric acid

dilution:
electrode buffer:

dilution: Cathode; 1:3 dH20

anode; 1:4 dH20
Ayala-C (Ayala, et al., 1972)

gel buffer:

electrode buffer:

dilution:

.009 M Tris

.003 M citric acid

pH 7.0

. 135 M Tris

.040 M citric acid

pH 7.0

TC-5.5 (Selander, et al
gel buffer:

dilution:

. 1971)

.064 M Tris

.026 M citric acid

pH 5.5

1:2 dH20

3. Continued.

electrode buffer;

dilution:

.223 M Tris

.093 M citric acid

pH 5.2

3:1 dH20

The buffer system used for each specific enzyme is
listed in Table 1. The electrophoretic run was terminated
when the bromphenol blue dye markers had migrated to the end
of the gel (8.5 cm). The 1 cm thick gel was removed from the
gel mold by making an incision through the leading edge, just
in front of the dye marker. The gel was then cut into five,
1.5 mm thick slices by placing the gel on a plexiglass guide
and using a .012 inch diameter guitar string mounted in a
hack saw frame. Each slice was then stained for a particular
enzyme.

Twenty enzyme systems, representing the products of
twenty-seven loci were assayed. The names and Enzyme
Commission numbers (E.C. No.) for each enzyme, as provided by
the Commission on Biochemical Nomenclature (1972) , are listed
in Table 1. The abbreviation listed will be used throughout
this report to indicate the enzyme system (all upper case
letters) or genetic locus (only first letter capitalized) .

The staining methods described below are those from
Steiner and Joslyn (1979) , unless otherwise noted. The
quantities listed were for 50 ml of staining solution, the
volume required to stain a 1.5 mm gel slice. The following

Table 1. Names, Enzyme Commission numbers, locus
designations and buffer system for the
enzymes assayed in this study.

Enzyme name

E.C.

No.

Abbrev.

Locus

Buffer
System

Acid phosphatase

3

1

3.2

ACPH

Acph

CA-5.5

Aconitase

4

2

1.3

ACON

Aeon

CA-7 . 0

Adenylate kinase

2

7

4.3

ADK

Adk

CA-7 . 0

Catalase

1

11.1.6

CAT

Cat

CA-8 . 0

Esterase

3

1

1.1

EST

Est-1
Est-2
Est-3
Est-4

CA-7 . 0
CA-7 . 0
CA-7.0
CA-7 . 0

Glutamate oxaloacetate
transaminase

2

6

1.1

GOT

Got-1

CA-8 . 0

alpha-Glycerophosphate
dehydrogenase

1

1

1.8

GPDH

Got-2

CA-8 . 0

Hexokinase

2

7

1.1

HK

Hk-1
Hk-2

CA-7 . 0
CA-7 . 0

Hydroxyacid

dehydrogenase

1

1

1.30

HAD

Had

CA-8 . 0

Isocitrate dehydrogenase

1

1

1.42

IDH

Idh-1
Idh-2

CA-8 . 0
CA-8.0

Lactate dehydrogenase

1

1

1.27

LDH

Ldh

CA-8 . 0

Malic dehydrogenase

1

1

1.37

MDH

Mdh

CA-8 . 0

Malic enzyme

1

1

1.40

ME

Me

CA-8 , 0

Mannose phosphate
isomerase

5

3

1.8

MPI

Mpi-1
Mpi-2

CA-7 . 0
CA-7 . 0

Peptidase

3

4

1.1

PEP

Pep

CA-8 . 0

Phosphoglucomutase

2

7

5.1

PGM

Pgm

CA-8 . 0

6-Phosphogluconate

1

1

1.43

6-PGD

6-Pgd

CA-5.5

dehydrogenase

Table 1 continued,

Enzyme name E.G. No. Abbrev. Locus Buffer

System

Phosphoglucose isomerase 5.3.1.9 PGI Pgi CA-8 . 0
Sorbitol dehydrogenase 1.1.1.14 SODH Sodh CA-8.0
Xanthine dehydrogenase 1.2.1.37 XDH Xdh CA-8.0

abbreviations are used: MTT ([3- (4, 5 Diinethylthiazol-2-yl) -
2 , 5-diphenlytetrazoliuin bromide]), NAD (nicotinamide adenine
dinucleotide) , NADP (Nicotinamide adenine dinucleotide
phosphate) , and PMS (phenazine methosulfate) .

All reagents were purchased from Sigma Chemical Co. , St.
Louis, Missouri.

1. ACPH acid phosphatase; sodium alpha naphthyl acid
phosphate, 50 mg; polyvinylpyrolidine, 100 mg; 0.1
M manganese chloride; 0.5 ml; sodium chloride, 500
mg; 0.05 M acetate buffer pH 5.0, 50 ml. After
incubating at 37°C for 3 0 minutes 50 mg of Fast
Blue RR was added.

2. ACON aconitase (Shaw and Prasad, 1970): cis-
aconitic acid, 60 mg; 0.1 M magnesium chloride,
0.5 ml, NADP, 10 mg; isocitrate dehydrogenase, 20
units; MTT. 10 mg; 0.2 M Tris-HCl pH 8.0, 50 ml
After incubation at 37°C for 30 minutes 5 mg of PMS
were added.

3. ADK adenylate kinase: glucose, 200 mg; adenosine
diphosphate, 40 mg; 0.1 M magnesium chloride, 5 ml;
NADP, 10 mg; glucose-6-phosphate dehydrogenase, 3 0
units; hexokinase, 60 units ; MTT, 10 mg; PMS, 5
mg; 0.2 M Tris-HCl pH 8.0, 50 ml.

4. CAT catalase (Shaw and Prasad, 1970): 35% hydrogen
peroxide, 0.1 ml; dH20, to 100 ml. Following
incubation at room temperature for 15 minutes the

10

solution was drained and the gel rinsed with water.
Solutions of 2% potassium ferricyanide, 25 ml and
2% ferric chloride, 2 5 ml were added and the
mixture agitated until white bands appeared on the
gel .

EST esterase: alpha-naphthyl acetate, 40 mg; beta-
naphthyl acetate, 2 0 mg; 0.2 M phosphate buffer pH

6.4, 50 ml. After incubating for 30 minutes 50 mg
of Fast Blue RR was added.

GOT qlutamate oxaloacetate transaminase: L-aspartic
acid, 400 mg; alpha-ketoglutaric acid, 185 mg;
pyridoxal-5-phosphate, 10 mg, 0.2 M Tris-HCl pH

8.5. After incubating for 30 minutes at 37 °C, 50 mg
of Fast Blue RR was added.

a-GPDH alpha-qlycerophosphate dehydrocfenase : alpha-

glycerophosphate, 50 mg; NAD , 2 0 mg; MTT, 10 mg;

0.2 M Tris-HCl pH 8.0, 50 ml. After incubating at

37 °C for 3 0 minutes, 5 mg of PMS were added.

HAD hydroxyacid dehydrocfenase: D-gluconic acid, 100

mg; 0.1 M magnesium chloride. 0.5 ml; sodium

chloride, 100 mg; 0.2 M Tris-HCl pH 8 . 0 , 50 ml;

After incubating at 37 °C for 3 0 minutes, 5 mg of

PMS were added.

HK hexokinase: glucose, 50 mg; adenosine

triphosphate, 4 0 mg; 0.1 M magnesium chloride, 1 ml;

NADP, 10 mg; glucose-6-phosphate dehydrogenase, 20

units; MTT, 10 mg; 0.1 M Tris-HCl pH 7 . 5 , 50 ml. After

11

incubating at 37° for 15 minutes, 5 mg of PMS were
added.

10. IDH isocitrate dehydrogenase; sodium isocitrate,
50 mg; 0.1 M magnesium chloride, 2 ml; NADP, 10 mg;
MTT, 10 mg; 0.1 m Tris-HCl pH 7.5, 50 ml. After
incubating for 15 minutes at 37 °C, 5 mg of PMS were
added.

11. LDH lactate dehydrogenase: lithium lactate, 300
mg; NAD, 20 mg; MTT, 10 mg; 0.2 M Tris-HCl 50 ml.
After incubating for 60 minutes at 37 °C, 5 mg of
PMS were added.

12. MDH malic dehydrogenase; 2.0 M DL-malate pH 7.0, 3
ml; NAD, 20 mg, MTT, 10 mg; 0.2 M Tris-HCl pH 8.0,
50 ml. After incubating at 37°C for 30 minutes, 5
mg of PMS were added.

13. ME malic enzyme; 2.0 M DL-malate pH 7.0, 2 ml; 0.1
M magnesium chloride, 2.5 ml; NADP, 10 mg; MTT, 10
mg; 0.1 M Tris-HCl pH 7 . 0 . 50 ml. After
incubating at 37 °C for 3 0 minutes, 5 mg of PMS were
added.

14. MPI mannose phosphate isomerase; (Harris and
Hopkins, 1976): mannose-6-phosphate, 20 mg; 0.1 M
magnesium chloride, 1 ml; NADP, 10 mg; glucose-6-
phosphate dehydrogenase, 2 0 units; phosphoglucose
isomerase, 2 0 units; MTT, 10 mg; 0.2 M Tris-HCl pH
8.0, 50 ml. After incubating at 37 °C for 30
minutes 5 mg of PMS were added.

12

15. PEP peptidase; L-leucyl-tyrosine, 20 mg;
peroxidase, 2 5 mg; amino acid oxidase, 3 0 mg; 0.1 M
Tris-HCl pH 7.5, 50 ml. After incubating at 37 °C
for 30 minutes, 20 mg of 0-dianosidine-HCl was
added.

16. PGM phosphocflucomutase : sodium glucose-1-
phosphate, 35 mg; glucose-1, 6-diphosphate, 0.45
mg, 0.1 M magnesium chloride, 4 ml; NADP, 10 mg;
glucose-6-phosphate dehydrogenase, 2 0 units; MTT,
10 mg; 0.1 M Tris-HCl pH 7.5, 50 ml. After
incubating at 37 °C for 15 minutes, 5 mg of PMS were
added.

17. 6-PGD 6-phosphoaluconate dehydrogenase : 6-
phosphogluconate, 50 mg; 0.1 M magnesium chloride.
0.5 ml; NADP, 10 mg; MTT, 10 mg 0.1 M Tris-HCl pH
7.5, 50 ml. After incubating at 37 °C for 15
minutes, 5 mg of PMS were added.

18. PGI phosphoqlucose isomerase: f ructose-6-phosphate,
10 mg; 0.1 M magnesium chloride, 4 ml; NADP, 10 mg;
glucose-6-phosphate dehydrogenase, 10 mg; MTT, 10
mg; PMS, 5 mg, 0.1 M Tris-HCl pH 7 . 5 , 50 ml.
After incubating at 37 °C for 15 minutes, 5 mg of
PMS were added.

19. SODH sorbitol dehydrogenase (Shaw and Prasad,
1970) : sorbitol, 250 mg; NAD 20 mg; MTT, 10 mg;
0.2 M Tris-HCl pH 8.0, 50 ml. After incubating at
37 °C for 4 5 minutes, 5 mg of PMS were added.

13

20. XDH xanthine dehydrogenase: hypoxanthine, 100 mg;
NAD, 20 mg; MTT, 10 mg; 0.2 M Tris-HCl pH 8.0, 50
ml. After incubating at 37°C for 30 minutes, 5 mg
of PMS were added.
The name and number for enzyme loci and alleles were
assigned as follows: The first letter of the locus name was
capitalized. The loci were numbered in order, with the locus
having the highest mobility as number one. A biochemical
marker strain, called Q2 , was developed by sub-culturing the
ORLANDO strain of A. quadrimaculatus . With the exception of
Got-2 and Mpi-2 , the Q2 strain was fixed for a single allele
at all the enzyme loci included in this study. Numbers were
assigned to each allele based on its mobility relative to
that of the allozyme found in the Q2 strain. Except for Idh-
2 and Mpi-1, the Q2 allozyme (designated as 100) represented
the allozyme most common in field populations. In the case
of Got-2 and Mpi-2 the allele with the highest frequency in
the Q2 strain was designated as 100.

Crosses to determine inheritance patterns of enzyme
phenotypes were achieved using the induced copulation
technique, as described by Baker et al., (1962). The Q2
strain was crossed to Y-^ individuals reared from a field
population located at Ginnie Springs, Florida. Both the
parents and F^^ progeny were electrophoresed and stained for
the various enzymes.

14

Results
Enzyme Phenotypes

Techniques for visualizing enzyme phenotypes needed to
be developed before more detailed genetic studies could be
undertaken. A variety of buffer systems were available for
enzyme separation (Steiner and Joslyn, 1979; Selander, et
al., 1971; Ayala et al., 1972). The systems used were
obtained empirically, depending on banding quality and
consistent reproducibility. The banding phenotypes are
illustrated in Figures 1, 2, 3, and 4. All polymorphic loci
are illustrated in these figures either by gels containing
the most common allele and variants found in a field
population (Figures 1 and 2) or by gels representing the
results of genetic crosses (Figures 3 and 4) . These
observations also provided information concerning enzyme
quaternary structure. Table 2 describes the number of bands
observed in putative heterozygotes representing individuals
from the field and/or heterozygotes resulting from genetic
crosses. Enzyme structure was inferred from the number of
bands present in heterozygotes (Harris and Hopkins, 1976) .
Nine of the enzymes (Aeon, Adk, Est-1. Est-2 , Est-3 . Hk-1, Hk-
2, Mpi-1, and Pgm) were identified as monomers which appeared
as one band in homozygotes and two bands in heterozygotes.
There were ten enzymes which appeared to be dimers (Got-1,
Got-2. a-Gpdh. Had. Idh-1. Idh-1. Mdh. Pgi. Sodh. and 2^dh) .
These were always presented as three-banded heterozygotes.
The three banded phenotype always presented as two homodimer

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23

Table 2 . Description of enzyme phenotypes and evidence for
polymeric structure of 11 of 20 loci in Anopheles
quadrimaculatus . NX = not investigated.

No. of electroinorphs in gel phenotype

Locus Maximum per Present in Inferred
individual heterozygotes structure
of genetic cross

Aeon

2

Adk

2

Est-1

2

Est-2

2

Est-3

2

Got-1

3

Got-2

3

a-Gpdh

3

HK-1

2

HK-2

2

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Idh-1

3

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3

Mdh

3

Me

5

MDi-1

2

Pcnn

2

Pgi

3

Sodh

3

Xdh

3

NI

NI

2

2

2

3

3

NI

NI

NI

NI

3

3

3

5

2

2

NI

NI

NI

monomer
monomer
monomer
monomer
monomer

dimer

dimer

dimer
monomer
monomer

dimer

dimer

dimer

dimer
tetramer
monomer
monomer

dimer

dimer

dimer

24

bands migrating to the same position on the gel as the
respective homozygotes and a denser, hybrid band at a
position between the two homodimers (see especially figure
3B) . One enzyme Me presented a five banded heterozygote
(Figure 2D) phenotype indicating a tetrameric structure.
Observations concerning enzyme structure are only inferences.
Definitive determinations require enzyme purification and
dissociation-reassociation studies .

Unusual and Epigenetic Effects

Some banding patterns were observed which either did not
appear consistently or which failed to give predicted results
when studied by genetic crossing. In every gel stained for
Adk two rows of bands appeared cathodally to the major Adk
bands (Figure IB) . By comparing these to gels containing the
same material and stained for HK, it was determined that
these bands represented the two HK loci (Figure 2C) . In
addition, a band located anodally to the Adk bands was
sometimes present (Figure IB) .

The most interesting effects were observed in three
enzymes. Had, Acph, and 6-Pqd and were related to blood
feeding in females. Figure 5 illustrates the effect of blood
feeding on the electrophoretic mobility of these three
enzymes. In Had the mobility and intensity of the band were
increased in females 24 hours after blood feeding (Figure
5A) . Mobility returned to normal by 72 hours after blood

Figure 5. The effect of blood feeding on the phenotype
of certain enzymes in Anopheles
quadrimaculatus . All individuals used were
from the Q2 strain and were genotypically
identical for the three enzyme loci
illustrated. The individuals in the gels
shown were treated as follows: individuals 1-
3 = non-bloodfed females; 4-6 =females 24
hours after taking a blood meal; 7-9 = 48
hours post blood meal; 10-12 = 72 hours post
blood meal; 13-15 = 96 hours post blood meal;
16-18 following oviposition; 19-21 = males.

A. Hydroxyacid dehydrogenase. Individuals 1-3
normal position of the Had^^*^ allele in non-
blood fed females, 4-9 increased mobility and
intensity of staining in females analyzed 24-
48 hours following a blood meal; 10-18 return
to normal mobility, but with increased
intensity by 72 hours following a blood-meal,
16-18 normal mobility with some smeariness in
females following oviposition, 19-21 normal
mobility and faint bonding in adult males.

B. Acid phosphatase. Individuals 1-3 normal
mobility for the Acph^'^^ allele in non-blood
fed females, 4-6 increased mobility and
staining intensity in females 24 hours after
taking a bloodmeal, 7-15 mobility increased
further and bands smeary in females 48-96
hours post-bloodmeal, 16-18 mobility
decreased, but not at normal position and
bands compact in females following
oviposition, 19-21 normal mobility and weak
banding in adult males.

C. 6-Phosphogluconate dehydrogenase.
Individuals 1-3 normal mobility of the 6-
pg^lOO allele in non-blood fed females, 4-6
increased mobility and staining intensity in
females 24 hours after taking bloodmeal, 7-15
increased mobility, stain intensity and
smeariness in females 48-96 hours post-
bloodmeal, 16-18 return to normal mobility
with increased staining intensity in females
following oviposition, 19-21 normal mobility
and weak staining in adult males.

12 3456789 101112 131415 161718 192021

HAD

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B

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6-PGD

27

feeding, although staining intensity was higher than in non-
bloodfed females. A similar effect was observed in 6-Pad
(Figure 5C) . In this case mobility and intensity increased
24 hours after blood feeding, by 48 hours mobility remained
higher, but the banding became more diffuse. The diffuse
banding persisted through 96 hours after blood feeding.
Following oviposition the banding pattern returned to normal,
but staining was still more intense. The most profound
effect was observed in Acph (Figure 5B) . The pattern was
similar to that observed in 6-Pad. Increased mobility at 24
hours after a bloodmeal with diffuse banding at 48-96 hours
post bloodmeal. However in this case the smeariness
disappeared after oviposition, but the mobility remained
higher than in females which never had a bloodmeal.
Discussion

Electrophoretic techniques for the analysis of twenty-
seven enzyme gene loci were developed. Results revealed
genetic variability at twenty of the twenty-seven loci.
Inheritance patterns were determined for nine of the
polymorphic loci.

Epigenetic effects on three loci (Acph, Had and 6-Pad)
were shown to be related to blood feeding in females. These
effects alter the mobility of these isozymes and should be
considered when interpreting electromorphs.

These techniques can now be applied to studies on
genetic mapping and population genetics of A.
quadrimaculatus .

CHAPTER II

EXPERIMENTAL HYBRIDIZATION OF GEOGRAPHIC STRAINS OF

Anopheles quadrimaculatus (Say)

Introduction

Species in the genus Anopheles commonly evolve without
developing conspicuous morphological differences. An
increasing number of sibling species are being described in
this genus from throughout the world, as documented by
numerous authors (see Discussion section, this paper) .

Hybridization studies have been widely used to establish
the true biological species status of suspected sibling
species (Davidson, 1964, Davidson and Hunt, 1973, Paterson et
al., 1963). In addition, hybridization experiments have been
used to assess the degree of relatedness between sibling and
morphologically distinct, but related species (Davidson et
al., 1967, Kitzmiller et al., 1967).

The first sibling species complex described in the genus
Anopheles, was the Anopheles maculipennis complex. The
complex contains both Palearctic (Old World) and Nearctic
(New World) species. The Palearctic members include the nine

29

sibling species, Anopheles atroparvus Van Thiel, A.
beklemishevi Stegnii and Kabanova, A. labranchiae Falleroni,
A. maculipennis Meigen, A. martinius Shingarev, A. melanoon
Hackett, A. messae Falleroni, A. sacharovi Farre and A.
sicaulti Roubaud (White, 1978) . The Nearctic members of the
complex are morphologically distinct and include A. aztecus
Hoffmann, A. earlei Vargus, A. freeborni Aitken, A.
occidentalis Dyar and Knab and A. quadrimaculatus Say
(Buonomini and Mariani 1953, Kitzmiller et al., 1967).
Kitzmiller (1977) used polytene banding patterns to place A.
cfuadrimaculatus in a separate group that included Anopheles
walkeri Theobald and Anopheles artopos Dyar and Knab.
However, Joslyn (1978) recorded only nonviable eggs from
crosses with those species. On the contrary, viable eggs
were obtained from crosses of A. quadrimaculatus to A.
freeborni and A. aztecus (Kitzmiller, et al., 1967). These
results indicate a closer relationship between A.
quadrimaculatus and members of the Maculipennis complex,
instead of A. atropos or A. walkeri.

To date no sibling species have been described in the
Nearctic Anopheline fauna. A number of the Nearctic species
have broad distributions, and given what is known about other
species in this genus, make ideal subjects for studies on the
genetics of speciation. The purpose of the present study was
to assess the degree of cross fertility among nine field
populations of A. quadrimaculatus . This species has a broad

30
geographic distribution, ranging over the entire eastern half
of the United States.

Materials and Methods

The mosquitoes used in matings were offspring of females
collected from the following sites: in Florida at Ginnie
Springs (GIN) , Gainesville at Kanapaha Botanical Gardens
(KBG) , and Lake Panasofkee (PAN) ; in Alabama, 41 miles west
of Auburn on 1-85 (AUB) , and Guntersville (GUN) ; in
Mississippi at Skene (SKE) ; in Arkansas at Stuttgart (ARK)
and Bebee (BEB) ; and in Louisiana at Lake Charles (LAC)
(Figure 6) .
Field Collections

Both sexes were collected from daytime resting sites,
e.g. treeholes, farm buildings, and boxes placed in wooded
areas. Adults were put in Savage cages (Savage and Lowe,
1971) and provided with a 10% sucrose solution. The cages
were placed in styrofoam ice chests containing a small amount
of ice in plastic bags to keep the mosquitoes cool and
humidified. The chests were then air-mailed or transported
by car back to the laboratory.
Laboratory Procedures

On arrival, adults were transferred to larger cages (1
meter square) . Gravid females from each collection were
transferred individually to 3 0 dram containers for
oviposition and non-blooded females were provided with
bloodmeals by placing a confined guinea pig in the cage
overnight. Blooded females were removed, held for ovarian

0)

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33
development and placed in containers for oviposition.
Frequently, females would not oviposit, even though they were
obviously gravid. Females were traumatized to induce
oviposition by tearing one wing from the thorax with a
jewelers forceps. Following oviposition, females were
removed and the eggs left in the containers to hatch, usually
1 to 2 days after they were laid. Newly hatched eggs were
infused with 1/2 ml. of a 2% aqueous suspension of 2 parts
Tetramin Baby "E" fish food and 1 part brewer's yeast.

One day after hatching the larvae were transferred to
large (40.6cm X 50.8cm X 7.6cm) plastic trays in 2 to 2.5
liters of tap water. Larvae from females collected from the
same site were pooled. Each tray contained about 3 00 larvae
and the larvae were fed daily on 2 0 ml of the mixture
described above. Larvae were reared at 27 °C and pupation
usually began within a week of hatching. Pupae were removed
from trays and placed in 8 oz. plastic cups half-filled with
tap water. The cups were capped with 1 pint cardboard ice
cream containers covered with a mesh lid that provided a
place for emerging adults to collect. Adults emerged about
3 6 hrs post-pupation. Newly emerged adults were removed,
sexed, and placed in Savage cages. The adults were
maintained at 25 "C and 70-80% RH and were provided with a 10%
sucrose solution. The numbers of F^ adults obtained from
field collected females were adequate for the completion of
all the crosses, so that further maintenance of stocks by

34

inbreeding was not necessary. The initial series of crosses
were all between the F^ adults and ORLANDO (ORL) , a standard
laboratory strain maintained over forty years. The ORL
strain served as the standard against which all field strains
were compared. All crosses were accomplished using a
modification of the induced mating technique of Baker et al.
(1962) . Females were held for 2 to 5 days prior to mating.
In order to avoid wasting time mating females which might
subsequently refuse to take a bloodmeal, females were
bloodfed on guinea pigs or humans immediately prior to
mating. Sterility in hybrid males was determined by
microscopic examination of the testes. The testes and the
distal portion of the vasa deferentia were dissected out and
transferred to a small drop of saline on a slide. A cover
slip was added and gentle pressure was applied. The
preparation was examined at 400x. Sterility could be
detected by the absence of normal spermatozoa, and could
usually be predicted by the gross appearance of the testes
which were greatly reduced in size in most sterile
individuals. Sterility in females was tested by crossing to
fertile males.

Development of strains of sibling species A and B.

Four strains from the AUB and KBG populations were
developed for further study. These strains were selected on
the basis of the fertility of the F^ progeny obtained in
crosses to the ORL strain (i.e., produced fertile or sterile

35

hybrid males) . A series of isofeitiale lines were established
from field collected females from the AUB and KBG sites. A
sample of F^ males from each line was mated to ORL females
and the remainder of the F^^'s were sib-mated. The adult
males produced from the crosses to ORL were scored for
fertility. Lines which produced fertile hybrid males were
pooled and maintained as the A-strains and those which
produced sterile hybrid males were combined to make the B
strains. Thus two pairs of sympatric lines, AUB-A and AUB-B
and KBG-A and KBG-B, were developed.

Results
Survey of Field Populations

The results from the first series of crosses, involving
matings between field strains and ORL mosquitoes, are
presented in Tables 3 and 4. These data represent only egg
batches which hatched. A significant number of females from
all crosses laid egg batches which failed to hatch. This
phenomenon is undoubtedly due, in part, to the use of the
induced mating technique since induced matings often result
in copulation without the transfer of sperm (Bryan, 1973) .
These infertile matings appear normal, but the females are
not inseminated and lay only infertile eggs.

Results revealed the presence of two types of
individuals from the field, designated type A and type B.
Type A individuals were genetically compatible with ORL, type
B individuals were incompatible with ORL. The populations

36

Table 3. The percentage hatch, sex ratio, percent

survival to adult stage and F^ male fertility
in Type A population cross-matings.

Cross Number Percent

female of egg Percent Total Percent survival to Male
and male batches hatch adults males adult stage fertility

CONTROL
ORL X ORL 5
BEB X ORL 9
ORL X BEB 9
ORL X GIN 8
GUN X ORL 11
ORL X GUN 10
LAC X ORL 10
ORL X LAC 16
ORL X PAN 8
SKE X ORL 7
ORL X SKE 8

85.4

796

49.4

77.9

+

88.7

1332

52.5

78.3

+

85.9

870

51.5

90.0

+

91.7

883

56.3

91.4

+

86.0

1698

52.3

86.7

+

82.8

1088

48.2

82.9

+

91.7

2320

51.8

92.3

+

85.3

1359

52.4

88.1

+

78.7

700

55.7

77.4

+

87.7

968

51.7

86.1

+

80.8

1118

48.2

82.4

+

37

Table 4. The percentage hatch, sex ratio, percent survival to adult stage
and F-i male fertility in Type A/B population cross-mat ings.

Cross Number Percent

female of egg Percent Total Percent survival to Male

X male batches hatch adults males adult stage fertility

A-ARK

X ORL

12

71.4

1132

51.9

81.3

+

ORL X

A-ARK

13

80.9

1530

51.0

75.3

+

B-ARK

X ORL

2

66.4

125

54.4

78.1

-

ORL X

B-ARK

12

74.5

799

27.8

55.5

-

ORL X

A-AUB

20

nd

2723

55.0

nd

+

B-AUB

X ORL

10

70.1

982

51.4

70.3

-

ORL X

B-AUB

12

71.3

526

12.2

34.9

-

A-KBG

X ORL

6

83.3

519

48.8

66.1

+

ORL X

A-KBG

7

85.8

643

50.2

63.0

+

ORL X

B-KBG

7

nd

447

21.3

nd

-

nd: Data not determined.

38

were divided into two groups: type A populations, comprised
entirely of type A individuals and type A/B populations,
which were made up of a mixture of type A and type B
individuals. Crosses were made between all of the field
populations and ORL but all possible reciprocal crosses were
not achieved.

BEB, GIN, GUN, LAC, PAN and SKE were type A populations.
When crossed to ORL the F-^ progeny were normal in every
respect and the results were consistent with the control ORL
X ORL crosses (Table 3) . Crosses between individuals from
these six populations and ORL produced families with high
hatch (78.7 - 91.7%), high survival to adult stage (77.4 -
92.3%), 1:1 sex ratio (% males = 48.2 - 56.3) and fertile
male progeny. The percent survival to adult stage was
generally higher in the hybrid F^^ than in the control; the
average for the ten hybrid crosses was 85.6% compared with
77.9% in the control. In outcrossing a longstanding colony
strain to field material one might expect increased vigor in
the F^ resulting from heterosis. The populations ARK, AUB
and KBG are type A/B populations. Two types of results were
obtained from matings to ORL. Some crosses were identical in
outcome to those from the A populations, while others
resulted in the production of sterile males in the F^^. These
data were grouped into A and B crosses, and are presented as
such in Table 4. Crosses in which B females were mated to
ORL males were completed for the ARK and AUB populations. In
these crosses, hatch was high, survival to adult stage was

39

high and sex ratio was normal. In both crosses all F]^ males
were sterile. The reciprocal cross, ORL female X B male was
done for the ARK, AUB and KBG populations. Hatch was high in
the ARK and AUB crosses, but was not recorded for the KBG
crosses. Percent survival to the adult stage was
significantly lower in the ORL X B-ARK and ORL X B-AUB
crosses than in the respective reciprocal crosses. This was
due to heavy mortality in the male pupae. Consequently, the
sex ratio in the F]^ was strongly distorted in favor of
females, 27.8% males in the ORL X B-ARK cross, 12.2% males in
the ORL X B-AUB cross and 21.3% males in the ORL X B-KBG
cross. The abnormal male pupae produced from these crosses
are illustrated in Figure 7. Typically, in these pupae the
wing buds lie outside the cephalothoracic capsule (Figure
7A) . The wing buds became swollen with water and presumably
disrupted the pupa's ability to maintain buoyancy. In some
pupae, the head and thoracic appendages as well as the wing
buds were free (Figure 7B) . In addition to these
abnormalities some of the male pupae had deformed genitalia.
Normally, the pupal genitalia lie in a genital pouch, and in
males this pouch is somewhat pointed and bifurcated distally,
with lobes being equal in size. In some of the hybrid male
pupae one or both lobes were not developed (Figure 7C) . Many
of the pupae which did survive through the pupal stage died
during eclosion. Figure 7D shows a typical case where the
pupa has freed its abdomen from the puparium, but was unable
to free its head and thorax. No abnormalities were observed

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42
among the female pupae. Without exception all the hybrid
males in each family from crosses between ORL and B males
were sterile. Figure 8 compares the gross appearance of the
normal and hybrid male reproductive systems. In normal
males, the testes were ovoid and larger than the accessory
glands (Figure 8A) . The appearance of the hybrid male
reproductive system varied considerably. In many cases the
testes were completely atrophied and no wider than the vasa
deferentia. On the other extreme, some hybrid males had
testes which were normal in size, but contained no normal
spermatozoa. Figure SB shows the reproductive system of a
hybrid male, the testes are smaller than the accessory
glands, and contained no sperm. Figure 9 shows the contents
of normal (9A) and hybrid (9B) testes. A large ball of
spermatozoa has been extruded through the vas deferens of the
normal testis (Figure 9A) . A few abnormal sperm with short
tails can be seen in the hybrid testis (Figure 9B) .

Hybridization of A and B Strains

A second series of crosses were undertaken using A and B
strains developed from both the AUB and KBG populations.
Crosses were done to define, more completely, the
relationship between type A and type B individuals. The
results from these crosses are presented in Tables 5 and 6.

Data from matings within each of the four strains were
collected to establish the integrity of each strain and to
provide data to which hybrid crosses could be compared. As

Figure 8. The male reproductive system of A. quadrimaculatus

A. Normal male reproductive system

B. The male reproductive system, showing reduced
testes from a hybrid produced by crossing ORL
female and type B male.

t = testis; a.g. = accessory gland; v.d. = vas
deferens

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Table 5. The percentage hatch from A and B strains
and cross-matings.

47

Cross
female
and male

Number of
egg batches

Total
eggs

Percent
hatch

AUB-A X AUB-A
AUB-B X AUB-B
AUB-A X ORL
ORL X AUB-A
AUB-B X ORL
ORL X AUB-B
AUB-A X AUB-B
AUB-B X AUB-A
KBG-A X KBG-A
KBG-B X KBG-B
KBG-A X KBG-B
KBG-B X KBG-A
AUB-B X KBG-B
KBG-B X AUB-B

5

12

7

7

13

6

12

15

2

9

9

9

8

4

1376
2030
1022
1136
2119

756
3087
3495

532
1592
1819
1790

927

813

79.8
73.9
83.1
89.1
78.4
92.7
78.6
90.9
97.2
94.9
81.9
90.0
66.2
73.9

48

Table 6. The sex ratio, percent survival to adult stage and
F^ male fertility in A and B strains and
cross-matings .

Cross Number of Percent

female first instar Total Percent survival to Male

and male larvae adults males adult stage fertility

AUB-A

X

AUB-A

1098

669

56.9

60.9

+

AUB-B

X

AUB-B

1501

1157

53.5

77.1

+

AUB-A

X

ORL

1230

760

51.1

74.4

+

ORL X

AUB-A

1275

806

52.5

71.0

+

AUB-B

X

ORL

1662

1388

49.8

92.2

-

ORL X

AUB-B

701

258

0

36.8

NA

AUB-A

X

AUB-B

2426

589

0

23.5

NA

AUB-B

X

AUB-A

3176

1387

48.4

43.7

-

KBG-A

X

KBG-A

517

371

45.3

71.8

+

KBG-B

X

KBG-B

1510

758

56.6

50.2

+

KBG-A

X

KBG-B

1489

692

40.8

39.7

-

KBG-B

X

KBG-A

1609

639

54.7

46.5

-

AUB-B

X

KBG-B

614

431

48.3

70.4

+

KBG-B

X

AUB-B

601

516

52.3

85.9

+

49

expected, the members of each strain were compatible among
themselves. As a second control, each of the AUB strains as
crossed to ORL. Both reciprocal crosses between AUB-A and
ORL resulted in progeny which were normal in every respect.
The AUB-B female X ORL male crosses gave results similar to
those obtained from the first series of crosses (Table 4) ,
that is, hatch and development of the F-^ progeny appeared
normal, but all F-l males were sterile. The reciprocal cross,
ORL female X AUB-B male, produced results that were different
from the initial crosses, in that in contrast to the initial
cross where heavy mortality of the F^ male pupae was observed
(sex ratio of 12.2% males) (Table 4), this time all F^ male
pupae died (Table 6) .

Crosses between the A and B strains of sympatric origin
were conducted. The cross AUB-A female X AUB-B male produced
F^'s with the same characteristics as those produced when
AUB-B males were mated to ORL. Hatch was high (78.6%), but
the % survival to adult was low (23.5%), and all of the F^
males died in the pupal stage. Results were different for
the KBG-A female X KBG-B male cross. Mortality in male pupae
was not pronounced; and therefore the sex ratio was closer to
normal (40.8% males). Survival to adult stage was also
higher (39.7%). In both the reciprocal crosses, AUB-B female
X AUB-A male and KBG-B female X KBG-A male produced progeny
which were normal in viability, and the sex ratio was normal;
but in both cases all F]^ males were sterile.

50

The final pair of reciprocal crosses between AUB-B and
KBG-B established that these two strains were compatible-
Progeny resulting from these crosses were comparable in every
respect to the control (ORL X ORL) . All F-^ males were
fertile.
Backcrosses

Three of the four possible backcross combinations were
performed, using the AUB strains (Table 7) . Hatch was lower
in the backcrosses than in the F^ crosses. In both the F^
(AUB-A female X AUB-B male) and the F^ (AUB-B female X AUB-A
male) backcrossed to AUB-A the % survival to adult stage was
comparable t the A female X B male crosses. Sex ratio was
skewed in favor of females, but to a lesser degree than
either of the crosses: AUB-B female X ORL male or AUB-B
female X AUB-A male. In the cross, F^ (AUB-A female X AUB-B
male) female X AUB-B male, % hatch was also lower than in the
F^ crosses, however % survival to the adult stage was
significantly lower. Sex ratio was also skewed in favor of
females. Sterility persisted through the backcross, and all
of the backcross males were sterile.

Hybridization in Nature

A X B hybrid males can be recognized by microscopic
examination of the testes (Figure 8B) . Using this technique,
it was possible to examine field collected males and
determine if they were A X B hybrids. Males were collected

51

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52

from the AUB and KBG sites, returned to the laboratory, and
the testes were dissected and scored for sterility. Results
are presented in Table 8. Of the 14 3 males from the AUB
site and 185 from the KBG site all were normal, indicating
that all were either A or B types.
Discussion

A variety of different techniques have been applied to
the study of speciation. However, many of the measurable
species differences studied contribute little or nothing to
reproductive isolation. Hybridization experiments are
designed to measure post-mating reproductive isolation
directly, and as such have been recognized as the best method
available for the study of genie incompatibility (Templeton,
1981) .

The hybridization experiments described in this study
began with the screening of nine geographic populations in an
attempt to detect genetic incompatibility. ORL served as a
standard to which all field strains were compared. This
strategy eliminated the need of making all possible crosses
between field strains. Sterility in F^^ males was observed
for some of the crosses between ORL and three of the field
strains, ARK, AUB, and KBG while other crosses between these
strains and ORL produced normal F^^ progeny. These results
established the existence of two sympatric sub-populations at
these sites. Crosses between ORL and the remaining six field
strains produced normal males in the F]^. It should be noted
that these results should not be interpreted as meaning that

53

Table 8. Survey of AUB and KBG populations for presence of
sterile, hybrid males.

Number of Number of Number of
Population males examined fertile males sterile males

AUB 143 143 0

KBG 185 185 0

54

these populations are conspecific with ORL or each other.
Under the conditions of these experiments pre-mating
isolation between any of these strains would not be detected,
nor would post-mating isolation between specific field
strains. Crosses between A and B strains developed from the
AUB and KBG populations further confirmed the existence of
the two types. The incompatibility between the AUB-A X AUB-B
and KBG-A X KBG-B crosses proves the existence at these
sites, of two reproductively isolated and sympatric
populations. Crosses between AUB-B and KBG-B produced normal
progeny suggesting that these two populations are
conspecific.

Backcrosses of hybrid females to A and B males resulted
in a significantly lower hatch than was observed in any of
the control matings. The fact that male sterility persists
through the backcross indicates that potential gene flow
through F^^ females is inhibited.

The examination of field collected males from both sites
provided no evidence of hybridization, indicating that some
form of pre-mating reproductive isolation separates the A and
B populations in nature.

Kitzmiller et al. (1967) used the results of
hybridization experiments to assess the degree of
relatedness between different species in the A. maculipennis
complex. Their results agreed well with phylogenetic
estimates based on chromosomal differences. Relatedness is
determined by the degree of genetic compatibility with

55

results ranging from failure of sperm to fertilize eggs to
the production of adults in a normal 1:1 ratio but with
varying degrees of sterility. The former case indicated a
distant relationship, the latter, a close relationship.
Applying these criteria to A. quadrimaculatus species A and
B, it can be seen that they represent two closely related
sibling species. In the cross, species A female X species B
male, adults were produced, but the sex ratio was usually
distorted in favor of females. Survival through the larval
stages was high, but heavy mortality of male pupae was
generally observed. In the reciprocal cross adults were
produced in a 1:1 sex ratio and survival through all stages
was high. All male progeny from both crosses were sterile.
Female progeny can be described as semi-sterile since when
backcrossed to species A or B, hybrid females produced
smaller egg batches and hatch was low. Sterility in male
progeny persisted in the backcrosses.

Relationships similar to that between A. cfuadrimaculatus
Species A and B have been described between other sibling
species in the genus Anopheles. Within the Palearctic
species of the A. maculipennis complex, several species show
relationships comparable to the one described here. The
cross, A. labranchiae X A. atroparvus , produced sterile males
and fertile females, but in this case male fertility was
recovered in the F^ backcross to A. atroparvus males.
Sterility in both sexes was observed in the F]^ produced from
matings between A. maculipennis and A. atroparvus. whereas

56

only male progeny were sterile in the cross A. subalpinus
Hackett and Lewis X A. gambiae. All possible crosses between
members of the A. gambiae Giles complex have been made

(Davidson, 1964, Davidson and Hunt, 1973) , Some crosses
produced only males. Without exception all of the males were
sterile, and when produced, the females were fertile.
Crosses between A. merus Doenitz females to A. gambiae s.s.
or A.arabiensis Patton males produced all male progeny.
Likewise, matings between A. melas Theobald females and A.
gambiae s.s or A. arabiensis males produced only males. On
the other hand, the crosses A. melas female X A. gambiae
species D males and A. gambiae species D females X A. merus
males produced sex ratios strongly in favor of females (25
and 16.7% males respectively). Mahon and Meithke (1982)
report the results of crosses between the three sibling
species of A. farauti Laveran. The relationship between
these three species parallels that between A.
quadrimaculatus A and B. All crosses between A. farauti
species 1, 2 and 3 produced sterile male progeny. Sex ratio
distortion in favor of females was observed for the crosses
A. farauti species No. 1 female X No. 3 male (5% males) and
A. farauti no. 3 female X No. 2 male (9% males) , but the
reciprocals of each produced normal sex ratios. The general
pattern of sterile males and fertile females in the F-^ have
been reported for a number of other species, including the
crosses A. balabacensis Baisas X A. dirus Peyton and Harrison

(Baima and Harrison 1980) and A. sinensis Wiedemann X A.

57

enqarensis Kanda and Oguma (Kanda and Oguma 1978) .
Unidirectional male sterility has been reported in A.
culicifaces Giles, where the cross A. culicifaces species A
females X species B males results in sterile male progeny but
the reciprocal produces fertile males (Miles 1981) . In A.
coustani Laveran species A and B a similar unidirectional
effect has been observed (Coetzee 1983) . In this case when
A. coustani species B is the female parent, the cross
produces sterile male progeny. However, the reciprocal cross
results in the production of non-viable eggs.

In conclusion, hybridization studies involving nine
geographic strains of A. quadrimaculatus revealed the
existence of two reproductively isolated sympatric
populations. No evidence of natural hybridization between
the two forms was found. These results support the
conclusion that A. quadrimaculatus actually exists as two
sibling species, provisionally designated A. quadrimaculatus
Species A and A. quadrimaculatus Species B.

CHAPTER III

ENZYME POLYMORPHISM AND GENETIC STRUCTURE OF POPULATIONS

OF Anopheles quadrimaculatus SPECIES A AND B

Introduction

The European Anopheles maculipennis complex stands as a
classic example of sibling species and is cited in almost
every written account of the sibling species phenomenon
(Mayr, 1963; 1969; 1982; Dobzhansky, 1970; White, 1973;
Wright, 1978) . The entire complex consists of fourteen
Holarctic species. Interestingly, the nine Palearctic
species are all morphologically identical, or nearly so
(sibling species) , whereas the Nearctic fauna was,
heretofore, thought to be made up of five, closely related,
but morphologically distinct species. Recently,
hybridization studies have revealed that A. quadrimaculatus
Say, one of the Nearctic species, actually consists of two
sibling species (Chapter II) . These studies demonstrated
that the two species exist sympatrically at three of nine
localities sampled.

When gene flow is restricted between two populations,
differences in the composition of alleles and in their

58

59

frequencies within each population may develop. Such
differences can be measured by determining the allelic
frequencies at a number of loci within each population and
comparing them. One way to accomplish this is to measure
allozyme frequencies.

The purpose of this study was to measure the genetic
variability of A. quadrimaculatus in the southeastern United
States, in an attempt to answer several questions. First,
how much genetic differentiation exists between the two
species and between local populations of each? Second, does
the pattern of genetic differences confirm the existence of
two species? Third, do allozyme phenotypes occur which can
be used to distinguish reliably the two species? Finally,
what inferences can be made concerning the phylogenetic
relationships between the two species?

Materials and Methods

Adult A. quadrimaculatus were collected from the same
nine sites that were sampled in the previous hybridization
experiments: in Florida at Ginnie Springs (GIN) , at Lake
Panasofkee (PAN) , (Gainesville) and Kanapaha Botanical
Gardens (KBG) , and at Lake Panasofkee (PAN) ; in Alabama, 41
miles west of Auburn on 1-85 (AUB) , and at Guntersville
(GUN) ; in Mississippi at Skene (SKE) ; in Arkansas at
Stuttgart (ARK) and Bebee (BEB) and in Louisiana at Lake
Charles (LAC, Figure 6, Chapter II). One additional site,
not sampled in the hybridization experiments, was included.

60
Lake Seminole in the Florida panhandle at the Florida-
Georgia-Alabama state line (Figure 6, Chapter II) . The
collecting techniques employed were identical to those
described in Chapter II. On return to the laboratory-
collections were sorted on a cold table and stored at -60°C
until prepared for electrophoresis. All mosquitoes included
in this study were field collected adults.

Determination of species by hybridization was achieved
by crossing field collected males to species A females (ORL
strain, see Chapter II) . If the resulting male progeny were
sterile, or no male progeny were produced the parental male
was identified as species B, if normal male progeny were
produced the male parent was identified as species A.

Electrophoretic techniques for 27 loci in 20 enzyme
systems were described in Chapter I. Of these, the following
2 0 loci were included in this study: Aconitase (ACON, 1
locus) , Adenylate kinase (ADK, 1 locus) , Catalase (CAT, 1
locus) , Glutamate oxaloacetate transaminase (GOT, 2 loci) ,
alpha-Glycerophosphate dehydrogenase (alpha-GPDH, 1 locus) ,
Hexokinase (HK, 2 loci) , Hydroxy acid dehydrogenase (HAD, 1
locus) , Isocitrate dehydrogenase (IDH, 2 loci) , Lactate
dehydrogenase (LDH, 1 locus) , Malic dehydrogenase (MDH, 1
locus) , Malic enzyme (ME, 1 locus) , Mannose phosphate
isomerase (MPI, 2 loci). Peptidase (PEP, 1 locus),
Phosphoglucose isomerase (PGI, 1 locus) , Phosphoglucomutase
(PGM, 1 locus) , and Sorbitol dehydrogenase (SODH, 1 locus) .
Techniques used for the visualization of the enzymes.

61
including buffer systems, staining procedures and locus and
allele nomenclature are described in Chapter I. In addition,
Chapter I contains a description of the genetic basis of
isozymes at nine loci. The banding patterns of the remaining
isozymes were consistent with a genetic interpretation and
agree with previously described phenotypes in other
Anopheline species. Thus banding phenotypes (electromorphs)
could be scored as genotypes. To insure identity of alleles
between populations, a series of gels were run with samples
representing each population run concurrently on the same gel
in combination with all other populations.

Analyses of allele frequency data were performed using
the BIOSYS-1 computer program of Swofford and Selander
(1981) .

Results

Initially, allele frequencies were calculated under the
assumption that A. quadrimaculatus consisted of a single,
randomly mating population at each of the ten sites sampled.
This assumption was tested by calculating chi-square tests
for goodness of fit to Hardy-Weinberg equilibrium for each
polymorphic locus. A locus was considered polymorphic if the
frequency of the most common allele did not exceed 0.95
(Ayala, et al., 1974). This definition was used throughout
this report, unless otherwise stated. Significant deviation
from Hardy-Weinberg equilibrium was observed in two of the
tests. Chi-square values for Idh-1 and Idh-2 were highly

62

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dehydrogenase) showing the positions of the
diagnostic alleles at the Idh-1 and Idh-2
loci. Q-2 = marker strain serving as
control ; AUB-A = adult males of species A
from the AUB site; AUB-B = adult males of
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Q-2 AUB-A

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+

65

significant at four (ARK, AUB, KBG and SEM) of the ten sites
(Table 9). In addition, Selander's (1970) D coefficient is
negative in each case, indicating that a deficiency of
heterozygotes exists. These data strongly suggest at least
two populations at these sites.

The populations at ARK, AUB and KBG were known to be
composed of the two sibling species, designated A.
quadrimaculatus Species A and B. Differences between the two
species in allele frequencies at the IDH loci would account
for the deficiency in heterozygotes. To demonstrate this,
individual field collected males were positively identified
by hybridization, electrophoresed and the gels stained for
IDH. A total of 84 individuals from the AUB site and 22 from
the KBG site were tested. The Species B males were fixed for
a single allele at both the Idh-1 and Idh-2 loci, but species
A was polymorphic at these loci. Using the genotypes at
these two loci it was possible to correctly identify 32
individuals as species A and 74 as species B.

Figure 10 illustrates a typical IDH zymogram comparing
the two species. The diagnostic value of the IDH loci was
calculated after Ayala and Powell (1972) . Using the genotype
at the Idh-1 locus, individuals could be correctly identified
as being species A or species B with a probability of 98.61%,
The Idh-2 locus provided correct identification at a
probability of 98.43%. Neither satisfies the definition of a
diagnostic locus, which has been defined by Ayala and Powell
(1972) as a locus which provides correct identification at a

66

probability of 99% or higher. When the two IDH loci are used
together, the probability of correct identification is
increased to 99.98%. Thus, a tool was provided for rapid
identification of individuals which could be grouped into
discrete populations whose genetic constitution could then be
defined and compared.

Chi-square tests for goodness of fit to Hardy-Weinberg
were repeated following grouping assuming that both species
represented single panmictic populations at each site. The
results for the species A populations are presented in Table
10; species B was, as mentioned, fixed for a single allele at
each locus. In two populations (SEM-A and AUB-A) the tests
indicate a significant departure from Hardy-Weinberg
expectations. Prior to grouping, the IDH genotypes departed
significantly from Hardy-Weinberg equilibrium, but after
grouping a close-fit to predicted genotypic frequencies was
apparent, except as noted for SEM-A and AUB-A.

Table 11 presents allele frequency data for the twenty
loci analyzed. Also included are the number of individuals
examined per locus for each population. Data for the
population of species B at the ARK site is not presented
because it was not possible to obtain an adequate sample size
representing this population.

Comparing populations of species A with those of species
B, the loci having the greatest differences in allele
frequencies were Idh-1 and Idh-2 ♦ At the Idh-1 locus the 100

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73
allele predominated in populations of species A, whereas
populations of species B were fixed for the 85 allele. A
similar pattern was presented for the Idh-2 locus. The 13 6
allele was most frequent in species A populations while
populations of species B were fixed for the 173 allele.

There were four loci at which both species shared the
most common allele, but differed at alleles with intermediate
frequencies. At the Got-2 locus both species shared the 38
allele, but the average frequency of this allele in species A
was .335 while its frequency in species B was .049. There
were eight alleles at the Mpi-1 locus with both species
sharing the frequent allele. In populations of species A the
87 allele occurred with a frequency of .372, whereas, in
species B its frequency was .159. The 100 allele was the
second most common allele in species B with a frequency of
.324, the frequency of this allele in species A was .109.
With the exception of ARK-A and AUB-A, the 114 allele at the
Pgm locus was the second most common allele in populations of
both species, but occurred at very different frequencies. In
species B the 114 allele occurred at a frequency of .219, but
has a frequency of only .064 in species A. Likewise at the
Me locus the 100 allele was the most common allele in both
species, with the 94 allele being the next most frequent,
(absent in KBG-B) with an average frequency of .082 in
species A, but only .011 in species B.

Polymorphisms existed at six loci which showed little
differentiation between the two species. These included

74

Aeon. Got-1. Had. Mdh. Mpi-2 and Pep. Eight loci: Adk. Cat.
Gpdh, Hk-ly Hk-2 , Ldh. Sodh, and Pqi were not polymorphic by
the 0.95 criterion.

Measures of genetic variability, including mean numbers
of alleles per locus, percent polymorphic loci and mean
heterozygosity are presented for each population in Table 12,
These results indicate that populations of species B are less
variable genetically than those of species A. The number of
electrophoretically detectable alleles, occurring at a
frequency of at least 1%, varied from one (Cat, Hk-1, Hk-2,
Ldh) to eight (Mpi-1) . The mean number of alleles per locus
over all populations averaged 2.9 for species A and 2.3 for
species B. Species A was polymorphic at 50.5% of the loci
studied and species B at only 31.7%. Species B had a lower
mean heterozygosity as well, with 10.3% of its genes, on
average, in the heterozygous condition, while species A had a
heterozygosity of 15.9%.

Estimates of genetic distance and similarity between
species and between populations within species, were made
using the I and D statistics as defined by Nei (1978) . I and
D values for all pairwise comparisons are presented in Table
13. The average distance (D) between local populations of
species A was .005 (+ .003) and between populations of
species B was .002 (+ .014). Genetic identity and distance
coefficients demonstrate a high degree of differentiation
between the two species relative to that of local populations
within species. Genetic distance is much higher between

75

Table 12. Genetic variability in populations of Anopheles
quadrimaculatus species A and B.

Mean sample

Mean number

Percentage

Mean

size per

alleles per

of loci

hetero-

Populat

ion locus

locus

polymorphic^

zygosity^

SPECIES A

ARK-A

152.1

2.7

0.127

(19.9)

(0.3)

50.0

(0.037)

AUB-A

96.3

2.8

0.180

(7.2)

(0.3)

60.0

(0.042)

BEE

113.2

2.8

0.143

(9.4)

(0.3)

50.0

(0.038)

GIN

109.4

2.8

0.162

(6.5)

(0.3)

45.0

(0.044)

GUN

112.8

2.9

0.137

(6.7)

(0.3)

55.0

(0.037)

KBG-A

107.9

2.8

0.159

(14.3)

(0.3)

50.0

(0.043)

LAC

118.0

2.8

0.162

(8.0)

(0.3)

50.0

(0.042)

PAN

167.8

2.9

0.155

(13.7)

(0.2)

45.0

(0.043)

SEM-A

151.0

3.4

0.175

(13.4)

(0.3)

55.0

(0.040)

SKE

116.2

2.8

0.148

(9.5)

(0.3)

45.0

(0.042)

MEAN

124.5

2.9
SPECIES B

50.5

0.159

AUB-B

134.6

2.5

0.103

(13.9)

(0.3)

40.0

(0.038)

KBG-B

96.6

2.3

0.102

(6.8)

(0.3)

25.0

(0.042)

SEM-B

53.0

2.2

0.105

(2.0)

(0.3)

30.0

(0.041)

MEAN

94.7

2.3

31.7

0.103

^0.95 criterion
*-*Hardy-Weinberg expected

76

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in

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s

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77

species at the same locality than among the same species at
different localities.

A cluster analysis of the matrix of D values, using the
unweighted pair-groups method with arithmetic averages
(UPGMA, Sneath and Sokal, 1973), produced the dendogram
illustrated in Figure 11, The dendogram had two clusters at
a distance level of .092. One cluster contained the three
populations of Species B, and the other ten populations of
Species A.

Discussion

The data from this study confirm the existence of two
sibling species in what was formerly known as the single
species, A. guadrimaculatus . Analysis of genotypic
frequencies at two IDH loci revealed a highly significant
deficiency of heterozygotes at four of the sites sampled.
This phenomenon, known as the "Wahlund effect" (Crow and
Kimura, 1970) , is interpreted as resulting when
reproductively isolated populations occur sympatrically and
are sampled as a single population. These data alone provide
very strong evidence for the existence of two species (Makela
and Richardson, 1977; Bullini and Coluzzi, 1982).

The presence of Species A individuals heterozygous at
the IDH loci suggested that a limited amount of gene flow
might occur between the two proposed species. In fact, it
was revealed that species B was fixed for a single allele at
both IDH loci and species A was polymorphic and included the

w

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80

same two alleles for which species B was fixed, albeit at
relatively low frequencies.

A comparison of populations of the two species yielded
an average value for the Nei's distance coefficient of 0.092
(+ .014). This value is much smaller than the value between
sibling species of Drosophila (D = 0.60) reported by Ayala
(1975) . Average genetic distance between mosquito sibling
species are generally lower than those found in Drosophila.
The values reported in the literature for various Anopheline
sibling species are summarized in Table 14. The average
genetic distance between those members of the Palearctic A.
maculipennis complex which have been studied (excluding A.
melanoon x A. sacharovi) is 0.183. This is substantially
larger than the value between A. quadrimaculatus species A
and species B. Low values for genetic distance have been
reported in the A. qambiae complex (eg. A. qambiae x A.
arabiensis. D = 0.070). Extremely low values for D have been
reported between some members of the A. marshallii complex
(eg. A. marshallii species A x species B, D = 0.045 and A.
marshallii species A x species C, D = 0.029). These values
are comparable to the genetic distance between local
populations of A. maculipennis, D = 0.032 (Bullini and
Coluzzi, 1982). Bullini and Coluzzi (1973) observed that low
values for genetic distance, as in the A. qambiae complex,
are associated with higher levels of chromosomal divergence.
They suggest that the speciation process in such groups is

81

Table 14. Genetic distance (D) between sibling species of mosquitoes in the
genus Anopheles.

SPECIES COMPARISON

REFERENCE

Anopheles maculipennis corrplex

A. messae x A. subalpinus 0.119

A. subalpinus x A. melanoon 0.154

A. subalpinus x A. maculipennis 0.162

A. melanoon x A. maculipennis 0.228

A. labranchiae x A. atroparvus 0.250

A. melanoon x A. sacharovi 0.526

Bullini and Coluzzi, 1982

Anopheles qambiae cortplex
A. qambiae x A. arabiensis

0.070

Bullini and Coluzzi, 1982

Anopheles marshallii conplex
A. marshallii sp. A x sp. B
A. marshallii sp. A x sp. C
A. marshallii sp. A x sp. E
A. marshallii sp. B x sp. C
A. marshallii sp. B x sp. E
A. marshallii sp. C x sp. E

Lambert, 1983

0.045
0.029
0.118
0.107
0.220
0.128

Anopheles cajadrimaculatus conplex
A. caiadrimaculatus sp. A x sp. B

0.092

82

different than in groups with higher genetic distances, and
lower levels of chromosomal divergence, as in members of the
European A. maculipennis complex (Bullini and Coluzzi,
19823). Templeton (1981), however points out that the
difficulty in such interpretations is the fact that it is
impossible to distinguish whether these differences are
responsible for the speciation event or are consequences of
evolution subsequent to speciation. Genetic distance values
may only indicate how recently a speciation event has
occurred, being smaller between species which have more
recently diverged (Avise, Smith and Ayala, 1975; Carson,
1976) .

Conclusions can be drawn regarding the speciation
process in this case. There are several facts revealed in
the data in this dissertation which suggest that A.
quadrimaculatus species A is the ancestral species and that
species B evolved from it via a founder event. The genetic
distance between the two species is small, relative to the
distance values reported between mosquito sibling species,
which generally range from 0.10 to 0.30 (Bullini and Coluzzi,
1982) . Low values for genetic distance have been observed
between even morphologically distinct species recently
separated by founder events (Sene and Carson, 1977) . These
results conflict with conventional thinking which would
predict large genetic distances resulting from a "genetic
revolution" (Mayr, 19 54) produced by the founder event.
Templeton (1980) suggests that founder events are more likely

83

to affect only a small number of genes, while the majority of
the genome is unaffected (Templeton, 1980a) . In fact, there
is evidence that enzyme coding loci are relatively
insensitive markers of speciation (Templeton, 1980b) .
Populations which go through a small bottleneck
experience a decline in genetic variability. The magnitude
of the reduction was thought to be substantial, with only a
small proportion of the original genetic variability left
(Mayr, 1963). Nei, et al. (1975) studied the problem
quantitatively and determined the loss of variability to be
much smaller when population size increases follow the
bottleneck. Table 12 summarizes the data on genetic
variability in species A and B. Species B is less variable
genetically than species A. Species A has a mean
heterozygosity of 15.9% whereas species B has a
heterozygosity of 10.3%. The difference is modest, but fits
the level of decline predicted by Nei, et al. (1975). The
reduction in heterozygosity associated with founder
populations is generally attributed to the loss of low
frequency alleles by drift. The allelic composition of
populations of species B lack many of the low frequency
alleles found in populations of species A. Whereas, with the
exception of one rare allele (Pep^^°, p = 0.004), populations
of species B contain no alleles not also present in
populations of species A.

84
In conclusion, the electrophoretic data confirm the
existence of a sibling species. The genetic composition of
this new species suggest that it evolved from the ancestral
population through a small bottleneck, the founding
population may have consisted of less than ten individuals.

CONCLUSIONS

In summary, the results of this study prove the
existence of a new sibling species of Anopheles
quadrimaculatus (Say) . This report represents the first
description of a sibling species in the Nearctic Anopheline
fauna. This discovery is consistent with findings from a
large number of workers that Anopheline species frequently
evolve without developing significant morphological
differences.

The proof given here, for the existence of the new
species, is two-fold. Hybridization experiments revealed
that three of the nine populations surveyed existed as two,
reproductively isolated, sympatric populations. Reproductive
isolation was determined by mating studies which identified
male hybrid sterility. Attempts at identifying naturally
occurring hybrids at two of the sites failed, indicating that
a pre-mating mechanism, probably behavioral, maintains
reproductive isolation between these two species. A survey
of allozymic variation at twenty gene loci produced data
which supported the existence of a sibling species complex.
At two loci, a significant deficiency of heterozygotes was
revealed in the same three populations identified as being
mixed in the hybridization experiments. The genotypes at

86
these two loci could be used in distinguishing individuals of
the two species. The two species were tentatively designated
A. quadrimaculatus Species A and B.

The patterns of the genetic makeup of each species were
compared and a hypothesis concerning the phylogenetic
relationship between them was made. The evidence indicated
that species A is the ancestral species and that species B
evolved from it through a founder event.

There are five species, in addition to A.
quadrimaculatus . which belong to the Nearctic branch of the
Anopheles maculipennis complex. The results of this study
indicate that each of the remaining four should be more
closely studied to determine if additional sibling species
exist in this interesting species group.

87

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population genetic approach. Ann. Rev. Ecol. Syst.
12:23-48.

White, G. B. 1978. Systematic reappraisal of the Anopheles
maculipennis complex. Mosq. Syst. 10:13-44.

White, M. J. D. 1973. Animal Cytology and Evolutionary
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Wright, S. 1978. Evolution and the Genetics of Populations
vol. 4 Variability Within and Among Natural
Populations. University of Chicago Press, Chicago.

Biographical Sketch

Gregory Charles Lanzaro was born on October 2, 1950 in
New York City, New York. He graduated from Kansas State
University in 1972, with the degree of Bachelor of Science.
After graduation he served as a high school teacher of
biology at Omaha, Nebraska and New Haven, Connecticut. In
1975 he enrolled in graduate school at the University of
Arizona, where he obtained a Master of Science degree in
Entomology in 1978. In 1980 he began work for the Doctor of
Philosophy degree at the University of Florida. He is an
active member in four national scientific societies. At
present he serves as Assistant Medical Entomologist in the
Department of Entomology of Mississippi State University.

92

I certify that I have read this study and that in ny opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy. «i.ion ror tne

Jatk A. SeawrightT^Shai:

A. Seawright, 'Chairman
Associate Professor of
Entomology and Hematology

I certify that I have read this study and that in my opinion it
?u?fnL^.;rT^'''^ standards of scholarly presentatiS^ and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy. a^-Lon lor tne

Sudhir K. Narang

Adjunct Associate pf'ofessor of

Entomology and Hematology

L«5^^^^^^*^ ^ **^^® ^^^^ ^^^ ^^"^^y an'i that in my opinion it
?S?fnL^«?S''^?^^'''^ standards of scholarly presentatiSJi 2nd is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.

j^fr-v-^jU u). UJil

Donald W. Hall

Professor of Entomology and

Hematology

L«S^^^^"^^^ ^ ^^""^ ^^""^ ^^^^ ^t^^y »"d t^at in my opinion it
funHLm^^JS''^?^''^^^ standards of scholarly presentation and is
S^iiL J^ f ' ^"^«°°P« and quality, as a dissertation for the
degree of Doctor of Philosophy.

Stanley C. Schank
Professor of Agronomy

This dissertation was submitted to the Graduate Faculty of the
College of Agriculture and to the Graduate School, and was
accepted as partial fulfillment of the requirements for the
degree of Doctor of Philosophy.

December 1986 J^^fiS^j^Mj^

Dean, (Allege of Agriculture

nwlUFRSITY OF FLORIDA

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3 1262 08553 5564